Proteins with L-asparagine aminohydrolase activity, commonly known as L-asparaginases, have successfully been used for the treatment of Acute Lymphoblastic Leukemia (ALL) in children for many years. ALL is the most common childhood malignant cancers (Avramis (2005) Clin. Pharmacokinet. 44, 367-393).
L-asparaginase has also been used to treat Hodgkin's disease, acute myelocytic Leukemia, acute myelomonocytic Leukemia, chronic lymphocytic Leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Kotzia (2007) J. Biotechnol. 127, 657-669). The anti-tumor activity of L-asparaginase is believed to be due to the inability or reduced ability of certain malignant cells to synthesize L-asparagine (Id). These malignant cells rely on an extracellular supply of L-asparagine. However, the L-asparaginase enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia, thereby depleting circulating pools of L-asparagine and killing tumor cells that cannot perform protein synthesis without L-asparagine (Id).
L-asparaginase from E. coli was the first enzyme drug used in ALL therapy and has been marketed as Elspar® in the United States or as KIDROLASE and L-asparaginase MEDAC in Europe. L-asparaginases have also been isolated from other microorganisms, e.g., an L-asparaginase protein from Erwinia chrysanthemi, named crisantaspase, that has been marketed as ERWINASE (Wriston (1985) Meth. Enzymol. 113, 608-618; Goward (1992) Bioseparation 2, 335-341). L-asparaginases from other species of Erwinia have also been identified, including, for example, Erwinia chrysanthemi 3937 (Genbank Accession No. AAS67028), Erwinia chrysanthemi NCPPB 1125 (Genbank Accession No. CAA31239), Erwinia carotovora (Genbank Accession No. AAP92666), and Erwinia carotovora subsp. artroseptica (Genbank Accession No. AAS67027). These Erwinia chrysanthemi L-asparaginases have about 91-98% amino acid sequence identity with each other, while the Erwinia carotovora L-asparaginases have approximately 75-77% amino acid sequence identity with the Erwinia chrysanthemi L-asparaginases (Kotzia (2007) J. Biotechnol. 127, 657-669).
L-asparaginases of bacterial origin have a high immunogenic and antigenic potential and frequently provoke adverse reactions ranging from mild allergic reaction to anaphylactic shock in sensitized patients (Wang (2003) Leukemia 17, 1583-1588). E. coli L-asparaginase is particularly immunogenic, with reports of the presence of anti-asparaginase antibodies to E. coli L-asparaginase following intravenous or intramuscular administration reaching as high as 78% in adults and 70% in children (Id).
L-asparaginases from Escherichia Coli and Erwinia chrysanthemi differ in their pharmacokinetic properties and have distinct immunogenic profiles, respectively (Klug Albertsen (2001) Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown that antibodies that developed after a treatment with L-asparaginase from E. coli do not cross react with L-Asparaginase from Erwinia (Wang (2003) Leukemia 17, 1583-1588). Thus, L-asparaginase from Erwinia (crisantaspase) has been used as a second line treatment of ALL in patients that react to E. coli L-asparaginase (Duval (2002) Blood 15, 2734-2739; Avramis (2005) Clin. Pharmacokinet. 44, 367-393).
In another attempt to reduce immunogenicity associated with administration of microbial L-asparaginases, an E. coli L-asparaginase has been developed that is modified with methoxy-polyethyleneglycol (mPEG) This so-called mPEG-L-asparaginase, or pegaspargase, marketed as ONCASPAR (Enzon Inc.), was first approved in the U.S. for second line treatment of ALL in 1994, and has been approved for first-line therapy of ALL in children and adults since 2006.
ONCASPAR is an E. coli L-asparaginase that has been modified at multiple lysine residues using 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S. Pat. No. 4,179,337). SS-PEG is a PEG reagent of the first generation that contains an unstable ester linkage that is sensitive to hydrolysis by enzymes or at slightly alkaline pH values (U.S. Pat. No. 4,670,417). These properties decrease both in vitro and in vivo stability and can impair drug safety.
Furthermore, it has been demonstrated that antibodies developed against L-asparaginase from E. coli will cross react with ONCASPAR (Wang (2003) Leukemia 17, 1583-1588). Even though these antibodies were not neutralizing, this finding clearly demonstrated the high potential for cross-hypersensitivity or cross-inactivation in vivo. Indeed, in one report 30-41% of children who received pegaspargase had an allergic reaction (Id).
In addition to outward allergic reactions, the problem of “silent hypersensitivity” was recently reported, whereby patients develop anti-asparaginase antibodies without showing any clinical evidence of a hypersensitivity reaction (Wang (2003) Leukemia 17, 1583-1588). This reaction can result in the formation of neutralizing antibodies to E. coli L-asparaginase and pegaspargase; however, these patients are not switched to Erwinia L-asparaginase because there are not outward signs of hypersensitivity, and therefore they receive a shorter duration of effective treatment (Holcenberg (2004) J. Pediatr. Hematol. Oncol. 26, 273-274).
Erwinia chrysanthemi L-asparaginase treatment is often used in the event of hypersensitivity to E. coli-derived L-asparaginases. However, it has been observed that as many as 30-50% of patients receiving Erwinia L-asparaginase are antibody-positive (Avramis (2005), Clin. Pharmacokinet. 44, 367-393). Moreover, because Erwinia chrysanthemi L-asparaginase has a shorter elimination half-life than the E. coli L-asparaginases, it must be administered more frequently (Id). In a study by Avramis et. al, Erwinia asparaginase was associated with inferior pharmacokinetic profiles (Avramis (2007), J. Pediatr. Hematol. Oncol. 29, 239-247). E. coli L-asparaginase and pegaspargase therefore have been the preferred first-line therapies for ALL over Erwinia L-asparaginase.
Numerous biopharmaceuticals have successfully been PEGylated and marketed for many years. However, in many cases, PEGylated biopharmaceuticals show significantly reduced activity compared to the unmodified biopharmaceutical. In the case of L-asparaginase from Erwinia carotovora, it has been observed that PEGylation reduced its in vitro activity to approximately 57% (Kuchumova (2007) Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 1, 230-232). The L-asparaginase from Erwinia carotovora has only about 75% homology to the Erwinia chrysanthemi L-asparaginasc (crisantaspace). For ONCASPAR it is also known that its in vitro activity is approximately 50% compared to the unmodified E. coli L-asparaginase.
Thus, the technical problem underlying the present invention is the provision of means and methods for treating cancer, such as leukemia or non-Hodgkin's lymphoma, that avoids the limitations and disadvantages of prior art therapies, particularly of some PEGylated asparaginases.
The technical problem is solved by provision of the embodiments characterized in the claims.